The goal of this work is the elucidation of the mechanisms controlling the rate of protein synthesis at the cytoplasmic level in mammalian cells, and the differences between these mechanisms in normal and malignant cells. We have demonstrated that upon nutrient deprivation (essential amino acid, glucose or serum) of the Ehrlich ascites tumor cell in suspension culture, one such mechanism modulates chain initiation by inhibiting the formation of the complex between the 40s ribosomal subunit and the Met-tRNAf.eIF-2.GTP ternary complex (i.e., the 40s initiation complex). We are now examining eIF-2 function. During the initiation reactions, GTP in the ternary complex is hydrolyzed and eIF-2 is released as, or rapidly forms, eIF-2.GDP. We have found a GDP/GTP exchange factor (GEF) which allows GTP to replace GDP in the binary complex. This is a new initiation factor, since without GEF the half-time of dissociation of the eIF-2.GDP complex is greater than 30 minutes. Phosphorylation of the alpha subunit of eIF-2 prevents the GEF-catalyzed GDP exchange and thus can stop eIF-2 cycling and chain initiation. We are now investigating the relationship between phosphorylation and the inhibition of protein synthesis during physiological stresses including nutrient deprivation and heat shock.